Abstract
Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.
Highlights
Intrinsic hepatic drug clearance influences drug bioavailability and exposure
Why did the compensation for cytochrome P450s (CYPs) amount not consistently improve the correspondence in absolute values (AFD) for Human liver microsomes (HLM)- and human hepatocytes (HH)-derived CLint? We further examined the correlations between CLint and the CYP concentrations for each of the five probe substrates
We investigated the influence of specific protein amounts and intracellular unbound drug concentration (Kpuu) on the CLint of prototypical probe substrates in HLM and HH
Summary
Intrinsic hepatic drug clearance influences drug bioavailability and exposure. To investigate this, in vitro models are often used during drug discovery and development. The two most commonly used models are isolated hepatocytes and liver microsomes.[1−9] Isolated hepatocytes are the gold standard because these cells capture most of the factors influencing hepatic intrinsic clearance (CLint). They are used in various configurations to quantify metabolic activity as well as uptake and efflux transport of drugs and metabolites.[10] liver microsomes are usually the first screening tool in studies of metabolic clearance because of their low cost and ease of access.[11] Microsomes are derived by subcellular fractionation, with enrichment of the endoplasmic reticulum (ER).[12] Many membrane-bound drug metabolizing enzymes are located in the ER, including cytochrome P450s (CYPs) and many UDPglucuronosyltransferases (UGTs). Different results are often obtained from the hepatocytes and microsomes,[1,2,7,13,14] but the reasons for these differences are not fully understood
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